**4. Genetic manipulation of non-** *Saccharomyces* **yeasts**

Despite the increasing relevance of non-conventional yeast in modern enology, there are few examples and tools of genetic manipulation for those yeasts. The targets of modification are shared with *S. cerevisiae* strains and usually are devoted to an organoleptic improvement. Recently, Badura and co-authors [77] developed a tool for the genetic modification of *Hanseniaspora uvarum*. In the past, *Hanseniaspora* populations have been regarded to be spoilage yeasts due to some strains produce large quantities of acetaldehyde, acetic acid, and ethyl acetate. However, *Hanseniaspora* wine strains have oenological benefits such as lower final ethanol levels and higher acetate and ethyl ester concentrations. In this study, authors used a traditional PCR-based technique for the disruption of the *HuATF1,* which encodes a putative alcohol acetyltransferase involved in acetate ester formation. This approach introduces the first steps in the development of gene modification tools of this yeast.

Some *Kluyveromyces marxianus* strains are able to ferment sugars in high temperature environments (up to 45°C) including grape juice [78]. This yeast is also in some commercial preparations of yeast to contribute flavor complexity. In a study published in 2014, *K. marxianus* BY25569 strain was evolved and genetically engineered for overproduction of 2-phenylethanol (2-PE) from glucose [79]. 2-PE confers "rose" and "floral" scents, almost non-existent but interesting in winemaking. *Kluyveromyces lactis* is a kind of non-*Saccharomyces* yeast that aims to solve the problem of low total acid and high pH in wine, due to its high lactate production. In this direction, *K. lactic* was genetically modified by introducing a heterologous L-lactate dehydrogenase gene (LDH) and deleting pyruvate decarboxylase gene KlPDC1 and/or the pyruvate dehydrogenase (PDH) E1 subunit gene [80, 81]. With these modifications, the central carbon flux of *K. lactis* was diverged from the production of ethanol to enhance lactate production. *K. lactis* was also metabolically engineered for L-ascorbic acid (vitamin C) production [82]. On the palate, the wines with added ascorbic acid were perceived as less oxidized, less ripe and fresher. To achieve this aim, GDP mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2), and L-galactose-1-phosphate phosphatase (VTC4) from *A. thaliana* were introduced in *K. lactis* CBS2359 strain.

*Pichia pastoris* has been described as one of the most popular and standard tools for the production of recombinant protein in molecular biology [83]. This fact can be exploited in the wine production field. For example, The EPG1–2 gene, which codes for an endopolygalacturonase in *K. marxianus* CECT1043, has been expressed in *P. pastoris* X33 strain [84]. The use of this endopolygalacturonase improves Albariño wine aroma, providing an increase of citric, balsamic, spicy and above all floral (violet and rose) aromas [85].

CRISPR-based genome-editing approaches have also been applied in many non-conventional yeasts. However, due to non-*Saccharomyces* species had been considered spoilage yeasts in wine fermentations, and CRISPR in wine yeasts still falls under the definition of GMOs of the European regulations, less progress has been

*Genetically Modified Yeasts in Wine Biotechnology DOI: http://dx.doi.org/10.5772/intechopen.98639*

made in the field of fermented foods and beverages. Therefore, in non-conventional yeasts CRISPR-Cas9 system has been applied mainly in the production of biofuels, chemicals, nutraceuticals, enzymes or recombinant proteins [86, 87]. In *Pichia pastoris* (syn. *Komagataella phaffii*), CRISPR-Cas9 system has been applied to improve its efficiency for the production off high-value pharmaceuticals [88]; in *Ogataea polymorpha*, a thermotolerant methylotrophic yeast, for the production of bioethanol [89] or for the introduction of all the genes necessary for the biosynthesis of resveratrol [90]. For biofuels and chemicals production in *Issatchenkia orientalis* [91]; in *Kluyveromyces marxianus* for its use as cell factory [92, 93]; in *Kluyveromyces marxianus* for the production of recombinant proteins [94, 95], or for integrating a synthetic muconic acid pathway [96]; in *Schizosaccharomyces pombe* [97]; in *Candida* species for the production of xylonic acid and ethanol [98] or for biosynthesis of β-carotene and its derivatives [99]; and in *Yarrowia lipolytica* for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications [100].

In a recent study, CRISPR-Cas9 system was applied in the AWRI2804 *Brettanomyces bruxellensis* strain [101]. This specie has been described as the principal spoilage yeast in the winemaking industry. From the enological point of view, *B. bruxellensis* is known for its high resistance to ethanol and ability to survive in low-nutrient, low-pH conditions, allowing for long-term proliferation in winemaking processes [102]. Using CRISPR-Cas9 in combination with gene transformation cassettes tailored for *B. bruxellensis*, the authors were able to delete *SSU1* genes (conferring sulfite tolerance) and provide the means for targeted gene deletion in this species.
